*2.9. Antibacterial Activity of CS/PVOH/HNT and CS/PVOH/TO@HNT Films*

Figure 10 depicts the petri dishes used for the antimicrobial activity measurements of CS, CS/PVOH, CS/PVOH/xHNT, and CS/PVOH/xTO@HNT films against *E. coli* and *Staphylococcus* bacteria. Table 4 presents the antibacterial activity of the developed films that were based on the CS/PVOH nano-reinforcement. Four foodborne pathogenic bacteria cultivations i.e., *Escherichia coli*, *Staphylococcus aureus*, *Salmonella enterica*, and *Listeria monocytogenes* were used to test the antibacterial capacity of all films. The clear zone's diameter around the tested films indicates the magnitude of the inhibition of the microorganisms' growth. The absence of a clear zone, which means zero value of diameter, entails the absence of an inhibitory zone. Moreover, in this work, the bacteria growth in the area of direct contact of film with the agar surface was also studied. *Gels* **2022**, *8*, x FOR PEER REVIEW 14 of 25

**Figure 10.** Petri dishes images of (**a**–**c**,**e**,**f**) CS, CS/PVOH, CS/PVOH/xHNT, and (**d**,**g**,**h**) bacteria growth. Finally, CS/PVOH/HNT films containing 15% thyme oil displayed the **Figure 10.** Petri dishes images of (**a**–**c**,**e**,**f**) CS, CS/PVOH, CS/PVOH/xHNT, and (**d**,**g**,**h**) CS/PVOH/xTO@HNT films against *E. coli, S. aureus, S. enterica*, and *L. monocytogenes*.

CS/PVOH/xTO@HNT films against *E. coli, S. aureus, S. enterica*, and *L. monocytogenes*.

**Inhibition <sup>a</sup> Contact <sup>b</sup> Inhibition <sup>a</sup> Contact <sup>b</sup> Inhibition <sup>a</sup> Contact <sup>b</sup> Inhibition <sup>a</sup> Contact <sup>b</sup>**



Inhibitory zone surrounding film discs measured in mm after the subtraction of the disc diameter (6 mm); <sup>b</sup>Contact area of film discs with the agar surface; (+) indicates bacterial growth in the area, (-) indicates no bacterial growth in the area; Results expressed as mean ± standard deviation (n = 3); Means in the same column baring same superscript numbers are significantly equal (*p* > 0.05).

All the incorporated CS films displayed antibacterial effectiveness. The noted inhibition of the bacteria growth seems to have a dependency on the HNT and thyme oil

By reviewing the results, it is obvious that the growth inhibition was amplified upon increasing the concentration of the nanostructures and the EO. The CS/PVOH/HNT (5%, 10%, 15%) films showed pronounced antibacterial activity against the tested bacteria.

Specifically, the CS/PVOH/15HNT film exhibited higher antibacterial activity against *E. coli*, *S. enterica*, *S. aureus*, and *L. monocytogenes* if compared to CS/PVOH/5HNT, and CS/PVOH/10HNT. The inhibitory clear zones were noticeably higher for CS/PVOH/HNT

The CS/PVOH/5TO@HNT film inhibited all the tested bacteria by formatting clear zones of 7.50 mm for *E. coli*, 7.00 mm for *S. enterica*, 9.00 mm for *S. aureus*, and 8.00 mm for

Increasing the thyme oil concentration, it was also enhanced the zone of inhibition of




**Film Material** *E. coli S. enterica S. aureus L. monocytogenes*


CS 0.00 - 0.00 - 0.00 - 0.00 <sup>9</sup> +

CS/20PVOH/10TO@HNT 7.80 ± 0.20 - 8.00 ± 0.00 - 9.50 ± 0.50 - 9.03 ± 0.45 - CS/20PVOH/15TO@HNT 8.00 ± 0.50 - 9.00 ± 0.87 - 10.00 ± 0.00 - 9.00 ± 0.50 -


films when thyme oil (TO) was incorporated.

*aureus*, and *L. monocytogenes*.

CS/20PVOH 3.50 ± 0.87 - 3.83 ± 0.76 5,6,8 - 4.47 ± 0.81 <sup>3</sup>

(TO) concentration.

*L. monocytogenes.*

CS/20PVOH/5HNT 4.83 ± 0.29 <sup>1</sup>

CS/20PVOH/10HNT 5.00 ± 0.00 <sup>1</sup>

CS/20PVOH/15HNT 7.00 ± 0.50 <sup>2</sup>

CS/20PVOH/5TO@HNT 7.50 ± 0.50 <sup>2</sup>

a


**Table 4.** Antibacterial activity of active films against food pathogenic bacteria *E. coli*, *S. enterica*, *S. aureus*, and *L. monocytogenes*.

a Inhibitory zone surrounding film discs measured in mm after the subtraction of the disc diameter (6 mm); <sup>b</sup> Contact area of film discs with the agar surface; (+) indicates bacterial growth in the area, (-) indicates no bacterial growth in the area; Results expressed as mean ± standard deviation (*n* = 3); Means in the same column baring same superscript numbers are significantly equal (*p* > 0.05).

The chitosan films CS/PVOH/5HNT, CS/PVOH/10HNT, CS/PVOH/15HNT, CS/PVOH/5TO@HNT, CS/PVOH/10TO@HNT, CS/PVOH/15TO@HNT were compared to pure CS and CS/PVOH films. Pure CS films inhibited the growth of all tested bacteria but only by direct contact; except *L. monocytogenes* where no antibacterial activity was observed either in the contact area or by the formation of clear surroundings zones.

Furthermore, the CS/PVOH film showed antibacterial activity by formatting a clear zone of 3.50 mm for *E. coli*, 3.83 mm for *S. enterica*, and, 4.47 mm for *S. aureus* while no antibacterial effect against *L. monocytogenes* was observed.

All the incorporated CS films displayed antibacterial effectiveness. The noted inhibition of the bacteria growth seems to have a dependency on the HNT and thyme oil (TO) concentration.

By reviewing the results, it is obvious that the growth inhibition was amplified upon increasing the concentration of the nanostructures and the EO. The CS/PVOH/HNT (5%, 10%, 15%) films showed pronounced antibacterial activity against the tested bacteria.

Specifically, the CS/PVOH/15HNT film exhibited higher antibacterial activity against *E. coli*, *S. enterica*, *S. aureus*, and *L. monocytogenes* if compared to CS/PVOH/5HNT, and CS/PVOH/10HNT. The inhibitory clear zones were noticeably higher for CS/PVOH/HNT films when thyme oil (TO) was incorporated.

The CS/PVOH/5TO@HNT film inhibited all the tested bacteria by formatting clear zones of 7.50 mm for *E. coli*, 7.00 mm for *S. enterica*, 9.00 mm for *S. aureus*, and 8.00 mm for *L. monocytogenes.*

Increasing the thyme oil concentration, it was also enhanced the zone of inhibition of bacteria growth. Finally, CS/PVOH/HNT films containing 15% thyme oil displayed the highest antibacterial activity, resulting in a clear zone formation of 8.00 mm for *E. coli*, 9.00 mm for *S. enterica*, 10.00 mm for *S. aureus*, and 9.00 mm for *L. monocytogenes.*

In all cases, the nano-enforcement films showed significant antibacterial activity against Gram-negative bacteria and slightly stronger activity against Gram-positive bacteria.

It is known that chitosan possesses important antibacterial activity against a wide spectrum of bacteria. This activity is ascribed to its cationic nature (positively charged ammonium (NH<sup>4</sup> + )) that interacts with the negatively charged compounds of the bacteria cell wall [55]. However, CS does not show any migrated inhibitory activity [56]. Bacterial cell wall barry a negative charge, therefore electrostatic interaction between bacteria and positively-charged clays such as HNT, under specific conditions (pH, ionic force) is probable [57]. In order for clay to exhibit antibacterial activity, it is crucial to have the ability to maintain metal ions in solution and to have also sufficient interlayer cation exchange capacities [58]. Theoretically, HNTs do not meet these criteria, however, the literature refers to a wide range of possible modes of action of HNTs against bacteria. Abhinayaa et al., 2019 found that HNT at a concentration of 2.5 mg mL−<sup>1</sup> , was able to inhibit the growth of the phytopathogenic bacteria *Agrobacterium tumifaciens* and *Xanthomonas oryzae*, while at lower

concentrations it was observed decreased bacteria growth rate and damages on the cell membrane. These results were probably attributed to the effect of siloxane groups of HNTs surface in combination with the production of reactive oxygen species [59]. Moreover, increased antibacterial activity has been observed after the modification of the HNT surface. The functionalized HNT displayed strong antibacterial activity against the food-borne bacteria, *L. monocytogenes*, and *E. coli* [60,61].

HNT has been also found to be toxic to *E. coli* and *Salmonella typhimurium*, especially as a result of light-dependent oxidative stress [59,62]. Several antibacterial compounds such as antibiotics, essential oils, antibacterial peptides, etc. have been loaded into the HNTs in order to enhance the antibacterial activity [63,64]. The HNT structure seems to allow the sustained release of the incorporated antibacterial agents such as thyme oil, leading to bacteria inhibition. By this controlled release, the thyme oil could enter the lipid layer of the bacterial cell wall causing cell death. Moreover, the interaction of bacterial cell walls with the thyme oil-HNT matrix may produce an oxidation-reduction response leading to cell death due to the production of reactive oxygen species [65].

Concluding, it is once more noted that in the present work, the bacteria growth was inhibited in a dose-dependent manner. The antibacterial efficacy of the tested films could be due to the nanocomposite films themselves but also might be due to the controlled release/migration of thyme oil. Many parameters play a role in the final antibacterial effect of a nanostructure, such as bacterial strain, nanoparticle type/size, chitosan molecular weight, growth media type, assay type, and bacterial cell concentration. Consequently, the increased antibacterial activity of the CS/PVOH/HNT films reported in this work is attributed to the synergistic effect of chitosan, HNT, thyme oil concentration, etc.
